Project summary/abstract As a normal aspect of animal development and homeostasis, programmed cell death (apoptosis) plays an essential role in maintaining the physiological balance of appropriate cell numbers by opposing uncontrolled cell proliferation. Abnormal inactivation or activation of apoptosis can lead to uncontrolled cell growth or uncontrolled cell death and may result in human diseases such as cancer, neurodegenerative diseases, and autoimmune disorders. The broad, long-term objective of this application is to understand the molecular mechanisms underlying the control and the execution of apoptosis and to use the knowledge from the study of apoptosis to facilitate development of new methods in treatment and prevention of human diseases caused by inappropriate cell death. Apoptosis is controlled and executed by an evolutionarily conserved cell death pathway. At the heart of this pathway is a family of highly specific "cell death" proteases, the caspases, which are first synthesized as inactive protease precursors and later activated through proteolysis during apoptosis. The activation of cell death proteases triggers systematic and orderly cell-disassembly of apoptotic cells and their removal by phagocytes. Several sensitized genetic screens, candidate-based reverse genetic screens, and biochemical screens have been carried out to identify CED-3 protease substrates and downstream components and pathways that trigger and coordinate the cell-disassembly and removal events. These studies have led to the identification of 20 new cell death genes and several candidate CED-3 substrates that act downstream of, or in parallel to, CED-3 to mediate various cell death execution processes, including chromosome fragmentation, mitochondria elimination, cell corpse recognition/engulfment, and other yet to be identified cell death signaling and execution events. The specific aims of this application are to: (1) characterize the functions of candidate CED-3 substrates in cell death execution;(2) identify and characterize additional genes involved in phosphatidylserine externalization, an "eat-me" signal, during apoptosis;(3) characterize genes involved in cell corpse recognition/engulfment and their acting mechanisms;and (4) characterize genes that affect other cell death execution processes. These studies should allow us to systematically identify molecular components and pathways involved in cell death execution and their functioning mechanisms. Some of the molecules identified in these studies may turn out to be potential targets for therapeutic drug designs in curing cancers or other human diseases caused by inappropriate apoptosis.